Analyses of Porphyria Caused by Environmental Pollutants : Concerted Reaction and Tempo of Heme and Porphyrin Syntheses

在真核生物中，当细胞面临紧急情况时，如氨基酸短缺，内质网应激和病毒感染，蛋白质合成终止。红细胞中的翻译起始受真核起始因子2a（真核起始因子2的a-亚基; eIF 2 α）激酶或HRI（血红素调节抑制剂）调节。在红细胞中，当细胞面临诸如血红素铁复合物短缺的紧急情况时，蛋白质合成被终止。HRI感测并介导对Fe（II）-原卟啉IX（血红素）浓度变化的反应。在正常条件下，当有足够的血红素时，HRI不会磷酸化eIF 2 α，但当血红素不足时，它通过磷酸化eIF 2 α来维持血红素与球蛋白的比例为1：1，从而启动蛋白质合成的终止。虽然HRI被报道为感测网织红细胞或红细胞中的血红素浓度，但其分子机制，特别是N-末端结构域在其寡聚化、激酶功能和血红素感测中的作用，仍然是难以捉摸的。此外，关于血红素如何结合到催化结构域并调节催化、血红素结合的化学计量以及哪些残基是血红素铁的轴向配体，知之甚少。在本研究中，我们试图阐明如何协同反应的血红素和蛋白质的合成耦合产生的N-末端截短的突变体，组氨酸突变体，半胱氨酸突变体和研究的催化功能，血红素亲和力，血红素配位结构。我们还研究了环境污染的重金属阳离子对HRI激酶活性的影响。结果表明，NO可增强HRI的催化作用，NO可恢复血红素和胆红素对HRI的抑制作用，重金属离子对HRI的催化作用有抑制作用。这些结果将有助于了解环境污染物引起卟啉病的分子机制。

In eukaryotes, protein synthesis is terminated when cells face an emergency such as a shortage of amino acids, stress to the endoplasmic reticulum, and viral infection. Translation initiation in the red cell is regulated by eukaryotic initiation factor 2a (a-subunit of eukaryotic initiation factor 2 ; eIF2α) kinases or HRI (heme-regulated inhibitor). In the red cell, protein synthesis is terminated when cells face an emergency such as shortage of the heme iron complex. HRI senses and mediates responses to changes in the Fe(II)-protoporphyrin IX (heme) concentration. Under normal conditions when there is sufficient heme, HRI does not phosphorylate eIF2α, but when there is a shortage of heme, it acts to maintain the heme-to-globin ratio at 1:1 by phosphorylating eIF2a, which initiates the termination of protein synthesis. Although HRI is reported to sense the heme concentration in reticulocytes or red blood cells the molecular mechanisms, in particular the role of the N-terminal domain in its oligomerization, kinase function, and heme sensing, have remained elusive. In addition, little is known regarding how heme binds to the catalytic domain and regulates catalysis, the stoichiometry of heme binding, and which residues are axial ligands for the heme iron. In the present study, we attempted to elucidate how the concerted reactions of the heme and protein syntheses are coupled by generating the N-terminal truncated mutants, His mutants, Cys mutants and examining the catalytic function, heme affinities, and heme coordination structures. We also examined effects of environmentally polluted heavy metal cations on the kinase activities of HRI. It was found that NO enhances catalysis of HRI, NO recovers inhibited calysis by heme and bilirubin and heavy metal cations inhibit catalysis. The results obtained will be helpful to understand the molecular mechanism of porphyria caused by environmental pollutants.